clazah and johntenpiece
Here is my explanation of the process of producing insulin (principles remain the same for other proteins/applications). Probably will be unlikely for VCAA to want all the specifics on the reporter gene and certain chemicals, but I find some of these details helpful when trying to understand the bigger picture of the process. Honestly, at this point, it's hard to really know what VCAA will want, considering we're all on a new study design.
Making the recombinant plasmids:
- Plasmid vectors are prepared by scientists, containing the ampR (ampicillin resistant gene -> antibiotic resistance). It also contains a reporter gene called lacZ and specific recognition sites specifically for BamHI. Note that when lacZ is expressed, if produces what is called β-galactosidase, which is an enzyme that converts a substrate called X-gal from colourless to blue.
- Two plasmids are required, one for insulin subunit A and one for insulin subunit B (insulin is a quaternary protein). BamHI is then used for both plasmid samples to make a cut in each plasmid and two cuts in each insulin subunit. This forms complementary sticky ends. Note that the cut in the plasmid occurs in the middle of the LacZ reporter gene.
- DNA ligase is then used to join the subunits into their respective plasmids by creating phosphodiester bonds (reforms the sugar-phosphate backbone), forming two different types of recombinant plasmids.
Creating the transformed bacteria:
- Plasmids are added to a solution of E.coli bacteria.
- Heat shock or electroporation is used to increase the permeability of the bacterial plasma membranes, encouraging them to take up the plasmids. Of course, not all the bacteria will
- Bacteria cultures are spread and incubated on agar plates containing X-gal and ampicillin. Colourless colonies can be determined as the transformed bacteria with the insulin subunit gene in it, as the lacZ gene is not longer active due to the insulin gene of interest having been inserted in the middle of it. Blue colonies are therefore concluded to be the transformed bacteria without the insulin subunit gene in it. Obviously, any colonies that are still surviving highlights that they contain the plasmids due to be ampicillin resistant (non-transformed bacteria will have died off).
- Recombinant plasmids then produce an insulin subunit with a β-galactosidase tail,
which is formed from the half of the lacZ gene which has been transcribed and translated (probably not all that important to know about).
Producing the insulin and extracting it:
- The transformed bacteria that contain the plasmids are placed in conditions that allow them to exponentially reproduce before their membranes break down.
- The insulin that they produce is isolated and purified.
- The two insulin chains have their β-galactosidase tails removed.
- The two insulin chains are then mixed, allowing them to form disulphide bonds (covalent bonds between sulphur atoms in the insulin subunits) to create functional human insulin that can be used for diabetic patients.
In terms of the general process, a similar process occurs, but different antibiotic resistance genes and reporter will be used. The restriction endonucleases will differ as well. Anyway, I hope this helps!
